This assay provides a robust method for measuring cytotoxicity in a readily automated 384-well format. Cell viability is determined using the CellTiter Glo reagent (Promega), which gives a luminescent readout of cellular ATP levels. ..more
BioActive Compound: 1
Description:
This assay provides a robust method for measuring cytotoxicity in a readily automated 384-well format. Cell viability is determined using the CellTiter Glo reagent (Promega), which gives a luminescent readout of cellular ATP levels.
Using this assay we have determined the cytotoxicity of doxorubicin against human pulmonary artery cells (HPAECs), which are a target of the aerosol delivery of therapeutic agents. Doxorubicin was provided spiked in 30 random locations in a 384-plate provided from Discovery Partners International (DPI) through the MLSCN.
Using this assay we have determined the cytotoxicity of doxorubicin against human pulmonary artery cells (HPAECs), which are a target of the aerosol delivery of therapeutic agents. Doxorubicin was provided spiked in 30 random locations in a 384-plate provided from Discovery Partners International (DPI) through the MLSCN.
Protocol
(1) Seed HPAECs in opaque clear-bottom 384-well plates (Corning) at 1250 cells/well in 20 uL media.
(2) After 24 h at 37 oC, add doxorubin in 20 uL media to give a final concentration of 2 uM. (Final DMSO concentration of 0.4 percent).
(3) Seal with gas-permeable membrane, and incubate for 72 h at 37 oC.
(4) Add 40 uL of CellTiter Glo and allow mixture to stand for at least 30 min.
(5) Read luminescence on Envision.
Data analysis:
A template written in ActivityBase (IDBS) automatically calculates percent inhibition from raw luminescence data using within-plate controls (no compound) and blanks (no cells).
(2) After 24 h at 37 oC, add doxorubin in 20 uL media to give a final concentration of 2 uM. (Final DMSO concentration of 0.4 percent).
(3) Seal with gas-permeable membrane, and incubate for 72 h at 37 oC.
(4) Add 40 uL of CellTiter Glo and allow mixture to stand for at least 30 min.
(5) Read luminescence on Envision.
Data analysis:
A template written in ActivityBase (IDBS) automatically calculates percent inhibition from raw luminescence data using within-plate controls (no compound) and blanks (no cells).
Result Definitions
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| TID | Name | Description | Histogram | Type | Unit | |
|---|---|---|---|---|---|---|
| Outcome | The BioAssay activity outcome | Outcome | ||||
| Score | The BioAssay activity ranking score |  | Integer | |||
| 1 | Percent inhibition | Percent inhibition at 2 uM compound. Defined as cell viability relative to viability of control cells cultured in the absence of compound. Cell viability determined by measurement of level of cellular ATP using CellTiter Glo luminescent ATP detection kit. | | Float | % | |
| 2 | Mean percent inhibition: plate 1 | Average of percent inhibition from 30 wells containing doxorubin. | | Float | % | |
| 3 | SD percent inhibition: plate 1 | Standard deviation of percent inhibition from 30 wells containing doxorubin. | | Float | ||
| 4 | Number of replicates | | Integer | |||
| 5 | Mean percent inhibition: plate 2 | Average of percent inhibition from 30 wells containing doxorubin. | | Float | % | |
| 6 | SD percent inhibition: plate 2 | Standard deviation of percent inhibition from 30 wells containing doxorubin. | | Float | ||
| 7 | Number of replicates | | Integer | |||
| 8 | Mean percent inhibition control wells: plate 1 | Average of percent inhibition from 290 wells containing DMSO diluted to 0.4 percent but no test compound. | | Float | % | |
| 9 | SD percent inhibition control wells: plate 1 | Standard deviation of percent inhibition from 290 wells containing DMSO diluted to 0.4 percent but no test compound. | | Float | ||
| 10 | Number of replicates | | Integer | |||
| 11 | Mean percent inhibition control wells: plate 2 | Average of percent inhibition from 290 wells containing DMSO diluted to 0.4 percent but no test compound. | | Float | % | |
| 12 | SD percent inhibition control wells: plate 2 | Standard deviation of percent inhibition from 290 wells containing DMSO diluted to 0.4 percent but no test compound. | | Float | ||
| 13 | Number of replicates | | Integer |
Data Table (Concise)
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